The chemical composition of floral scents has been extensively investigated for hundreds of years because of the commercial value of floral volatiles in the perfumery industry. These investigations have determined that floral scents are almost always a complex mixture of small (approximately 100–250 daltons) volatile molecules and are dominated by monoterpenoid and sesquiterpenoid, phenylpropanoid, and benzenoid compounds. Fatty acid-derivatives and a range of other chemicals, especially those containing nitrogen or sulfur, are also sometimes present (for review, see Knudsen et al., 1993, Phytochemistry 333:253–280). However, in contrast to the chemical emphasis of the perfimers, until recently, there have been few studies concerning the biochemical synthesis of floral scent compounds and the enzymes and genes that control these processes. In fact, very recent investigations into the biogenesis of floral scent production in Clarkia breweri, an annual plant native to California, represents the best example to date in which isolation of enzymes and genes involved in the de novo synthesis of scent compounds in the flower have been reported.
Many plants emit floral scents, and such scents can attract a variety of animal pollinators, mostly insects. Floral scents vary widely among species in terms of the number, identity, and relative amounts of constituent volatile compounds. Plants did not naturally evolve to produce their scent for the benefit of humans; nevertheless, it is clear that humans find an aesthetic value in certain types of floral scents, and the presence of floral scent may have contributed to the decision by humans to cultivate and propagate specific plant species. While there is certainly a wide variation in human preference, most people prefer the scents of bee-pollinated and, especially, moth-pollinated flowers, which they often describe as “sweet-smelling”. Some volatile compounds found in floral scent have important functions in vegetative processes as well. They may function as attractants for the natural predators of herbivores or as airborne signals that activate disease resistance via the expression of defense-related genes in neighboring plants and in the healthy tissues of infected plants (Shulaev et al., 1997, Nature 385:718–721). They may also serve as repellents against herbivores (Gershenzon and Croteau, 1991, in Rosenthal, G. A. and Berenbaum, M. R., eds., Herbivores: Their Interactions with Secondary Plant Metabolites, 168–220). However, it cannot be taken for granted that the biosynthesis of such compounds in vegetative tissue will in all cases be identical (i.e., same reactions, same enzymes) to their synthesis in flowers.
Ornamental plants are valued for their visual attributes such as flower color and architecture, and plant habit. However, their non-visual benefits are also be deemed to be quite commercially valuable since these features might include unusual textures, but especially the fragrant volatiles emitted by both flower petals and foliage. Unfortunately, very few plants are currently cultivated primarily for their scent. It is a commonly-held perception today that intensely-bred, modern varieties of flowers have lost their ability to produce and emit floral scent. Consumers often raise this complaint when purchasing flowering plants with which they have strong expectant associations with their floral scent characteristics (such as roses and carnations). This perception, correct or not, has been attributed to the idea that a large number of commercial flower varieties have lost their scent during the selection and breeding processes due to, on the one hand, a focus on maximizing post-harvest shelf-life, shipping characteristics, and visual aesthetic values (i.e., expansive color offerings, shape, free-flowering characteristics), and on the other hand, to the lack of selection for the scent trait. While not rigorously tested or examined, plant breeders have long viewed the biochemical processes of floral scent production as energy-intensive, and which if minimized or eliminated, would conserve the plant's energy resources for the production of more flowers and/or longer-lasting blooms. This is especially unfortunate as the sensory experience associated with floral scent is currently in strong demand by the consumer.
In an attempt to satisfy the consumer's demand for floral scent, there are several possibilities that can be considered. In one approach, older ornamental varieties that have been characterized as fragrant could be re-introduced into the marketplace. In many instances, however, the horticultural performance of these older varieties may disappoint the consumer as they may not compare well with today's modern varieties (e.g., they may have short-lived flowers). This may seriously limit their consumer appeal and likelihood of commercial success. In other cases, these older, so-called ‘heirloom varieties’ may no longer be in cultivation and the floral scent-associated genes residing in the germplasm of these plants may be unrecoverable.
In an attempt to impart fragrance to cut flower stems or even intact plants, a number of ideas have been advanced which rely upon exogenous applications of fragrant, volatile molecules. Occasionally, fragrances are added back to a cut flower arrangement by way of commercially-synthesized fragrances that are sprayed onto the flower arrangement. Many of such fragrances are supported in an alcohol-based carrier that evaporates upon application, allowing the fragrance to permeate back into the air over a limited time. Commonly, however, such fragrances are lost two to three days after application, although the appearance of the flowers may continue for seven to fourteen days, before wilting occurs.
A series of patents describe devices for imparting fragrance to flowering plants or cut flowers. U.S. Pat. No. 4,827,663 (Stern) describes a flower arrangement apparatus, and in particular, an improved stem support including an encapsulated stem-sustaining plant oil mixture whereby the cut flower's fragrance can be maintained commensurate with the life of the flower arrangement itself. In this invention, an improved cut stem support in conjunction with a water-dissolvable capsule composed of a cut flower-sustaining plant oil mixture is described. The additive oil mixture is thought to float on the water and over time be absorbed into the flowers. An improved fragrance is thus obtainable from the floating oils themselves, as well as the petal of the flowers, which are imagined to permeate still additional fragrance through ongoing cellular activities. However, Stern nowhere demonstrates that the various plant oil mixtures that are described are actually taken up by the plants (nor demonstrates that these fragrances are even capable of being affected by ongoing cellular activities) and later emitted from the plant cell surfaces.
In a related invention, U.S. Pat. No. 5,353,546 (Bock) teaches a combination vase and air fragrance dispenser comprising two vessels, one for holding natural or artificial flowers, the other for holding a fragrance-emitting material. The two-vessel construction ensures complete separation between the flower and air treatment material, preventing contamination of the flower. The flower holding vessel is capable of receiving water needed to keep natural flowers fresh.
Similarly, U.S. Pat. No. 5,477,640 (Holtkamp, Jr.) teaches a fragrance-emitting plant watering system, wherein a potted natural flowering plant is seated within a larger vase-like solid fragrance emitter. A wick transports water from a water reservoir to a potted plant. An air freshener cartridge for emitting a fragrance is provided in a separate compartment of the device.
Finally, U.S. Pat. No. 6,013,524 (Friars et al.) describes a ‘living air freshener’ comprising a dwarf flowering plant such as a miniature rose plant rooted in a transparent or non-transparent growth medium in a transparent vessel, with a natural or artificial fragrance composition added directly to the growth medium or to a second compartment in said vessel. This invention provides a living air freshener that offers both an attractive flower display and a natural or artificial air freshening fragrance. Unlike cut flowers, it is envisioned that this product will actually grow, flower and die providing both air freshening and an attractive flower display. Like U.S. Pat. Nos. 5,353,546 and 5,477,640, this invention teaches that the aromatic compounds can be natural or artificial which are chemically inert (i.e., non-utilizable) to the plant, such that the compounds can be added directly to the growth medium or to a separate chamber or compartment of the display vessel in case that the aromatic compound is adversely affected by periodic watering of the plant, or the chemistry of the aromatic compound is adverse to the plant roots.
Collectively, these patents teach methods to construct an apparatus for enhancing the fragrance of either cut flowers or potted plants in which fragrance compounds are supplied exogenously to the plant tissues. In a somewhat-related example, U.S. Pat. No. 5,635,443 (Lesenko) describes a composition for enhancing the fragrance of cut flowers by providing (a) at least one surfactant, (b) at least one fragrance, (c) at least one fragrance solvent, (d) water and other lesser components like sodium chloride, sodium bicarbonate and an antifoaming agent in a liquid composition. The inventor speculates that the fragrance compound would then be taken up through the cut end of the stem of a cut flower, transported to the petal tissue and emitted from the flower. However, U.S. Pat. No. 5,635,443, like U.S. Pat. No. 4,827,663, does not teach whether the fragrance supplied to the cut flower is actually emitted from the flower or foliage. Moreover, if fragrance is detected, the inventor does not address the possibility that the fragrance may be due to direct volatilization of the compound from the vase water, and not emitted from plant tissues (as is contemplated in U.S. Pat. No. 4,827,663 and others noted above).
Taken together, U.S. Pat. Nos. 4,827,663, 5,353,546, 5,477,640, 6,013,524, and 5,635,443 describe devices and compositions for imparting natural or artificial fragrances to cut flowers and flowering plants. However, these methods do not teach how to create, maintain, enhance or modify floral scent using the natural cellular activities of the plants to synthesize and emit floral scent. That is, these patents describe the addition of already-scented, often-synthetic, volatile fragrance molecules to liquid or semi-solid compositions for fragrance emission. In essence, these methods describe artificial fragrance dispensers that emit the fragrance of plant oils extracted from the flowers, foliage or other plant parts. In contrast, the invention described herein describes a composition and methods on how to create, maintain, enhance or modify floral scent by treatment of a cut flower or flowering plant not with a naturally-occurring or synthetic fragrance compounds but with a floral scent precursor molecule which is able to be converted to a floral scent molecule by the metabolic activities of the plant cells (bioconversion), or which is able to stimulate the emission of natural floral scent components from the plant. That is, the floral scent emission pattern of the plant is specifically modified through the metabolic engineering of floral scent biosynthetic pathway(s) by exogenous applications of floral scent precursor compounds.
As noted above, in recent years, biochemists and molecular biologists have begun to address the biochemical questions surrounding floral scent biosynthesis and emission, largely in model organisms like Clarkia breweri and, to a much lesser extent, Antirrhinum majus L. (or snapdragon). Flowers of Clarkia breweri ([Gray] Greene; Onagraceae) an annual plant native to California, emit a strong sweet fragrance consisting of 8 to 12 different volatiles. These volatiles are derived from two biochemical pathways, one leading to monoterpenoids, and the other to phenylpropanoids. In the former group, one is linalool. In the latter group three are the volatiles (iso)methyleugenol, benzylacetate, and methylsalicylate. In addition, the formation of methylbenzoate, another phenylpropanoid, in snapdragon flowers has been very recently reported (Bushue et al., 1999, in Plant Biology '99, American Society of Plant Physiologists, p. 80).
Terpenes, especially monoterpenes such as linalool, limonene, myrcene, and trans-ocimene, but also some sesquiterpenes such as farnesene, nerolidol, and caryophyllene, are common constituents of floral scent. They are also often found in vegetative tissues, where they serve mostly as defense compounds. In work done mostly with vegetative tissue, but also with daffodil petals, it was found that monoterpenes are synthesized in the plastidic compartment. In this cellular compartment, isopentenyl pyrophosphate (IPP) is derived from the mevalonate-independent “Rohmer” pathway (Lichtenthaler et al., 1997, Plant Physiology 101:643–652.). IPP can be isomerized to dimethylallyl diphosphate (DMAPP), and one molecule of IPP is condensed with one molecule of DMAPP in a reaction catalyzed by the enzyme geranyl pyrophosphate synthase (GPPS) to form geranyl pyrophosphate (GPP), the universal precursor of all the monoterpenes. Similar work with vegetative tissue has revealed that in the cytosol, IPP is derived from the mevalonic acid pathway (McCaskili and Croteau, 1998, Trends in Biotechnology 16:349–355), and two molecules of IPP and one molecule of DMAPP are condensed in a reaction catalyzed by the enzyme farnesyl pyrophosphate synthase (FPPS) to form farnesyl pyrophosphate (FPP), the universal precursor of all the sesquiterpenes (McGarvey and Croteau, 1995, Plant Cell 7:1015–1026).
The phenylpropanoids, which are derived from the amino acid, phenylalanine, constitute a large class of secondary metabolites in plants. Many are intermediates in the synthesis of structural cell components (e.g., lignin), pigments (e.g., anthocyanins), and defense compounds. These are not usually volatile. However, several phenylpropanoids whose carboxyl group at C9 is reduced (to either the aldehyde, alcohol, or alkane/alkene) and/or which contain alkyl additions to the hydroxyl groups of the benzyl ring or to the carboxyl group (i.e., ethers and esters) are volatiles.
Work with C. breweri flowers has now resulted in the identification and characterization of four enzymes that catalyze the formation of four individual floral volatiles: linalool, (iso)methyleugenol, benzylacetate, and methylsalicylate. The enzymes are, respectively, linalool synthase (LIS), S-adenosyl-L-Met:(iso) eugenol O-methyltransferase (IEMT), acetyl-CoA:benzylalcohol acetyltransferase (BEAT), and S-adenosyl-L-Met:salicylic acid carboxyl methyltransferase (SAMT) (Dudareva et al., 1996, Plant Cell 8:1137–1148; Wang et al., 1997, Plant Physiology 114:213–221; Dudareva et al., 1998, Plant Journal 14:297–304; Dudareva et al., 1998, Plant Physiology 116:599–604; Wang and Pichersky, 1998, Archives of Biochemistry and Biophysics 349:153–160; Ross et al., 1999, Archives of Biochemistry and Biophysics 367:9–16). While IEMT and SAMT have relatively strict preferences for the substrates that they utilize [(iso)eugenol and salicylic acid, respectively], BEAT has been shown to utilize benzyl alcohol preferentially, but will also utilize other substrates like cinnamylalcohol and 2-napthaleneethanol very efficiently, to synthesize an array of aromatic compounds. LIS, like other monoterpene synthases, strictly utilizes GPP. Taken together, these results have established a substrate-product relationship for the bioconversion of non-fragrant floral scent precursors to fragrant floral scent components by the plant's enzymatic activities.
In C. breweri flowers, emission of the bulk of the volatiles occurs from the petals. Identification of the enzymes responsible for the formation of these volatile compounds has permitted investigators to determine how the levels of enzymatic activities are distributed in different floral parts and how they vary during flower development. When activity levels are calculated per total weight of each organ, the highest levels of activity of all these enzymes are found in the petals (Dudareva et al., 1998, Plant Physiology 116:559–604). Other parts of the C. breweri flower, however, also contain detectable levels of activity, and the stigma actually contains higher levels of LIS specific activity (but because the mass of the stigma of C. breweri is so small compared with the mass of the petals, LIS in the petal still comprises the majority of activity present in the flower). The specific types of cells expressing the genes encoding LIS and IEMT were determined by in situ hybridization. The results indicate that in C. breweri flowers, these scent genes are expressed uniformly and almost exclusively in cells of the epidermal layer of petals and other floral parts (Dudareva et al., 1996, Plant Cell 8:1137–1148; Dudareva and Pichersky, 2000, Plant Physiology 122:627–633). Volatile compounds produced in epidermal cells can apparently escape directly into the atmosphere after being synthesized.
Throughout the lifespan of the flower, the activities of LIS, IEMT, SAMT and BEAT in C. breweri follow complex patterns. C. breweri flowers do not show marked differences in emission between day and night. C. breweri flowers follow a long-term pattern in which emission peaks within a few days of anthesis and then declines gradually. In C. breweri, the activities of scent enzymes follow two different patterns. The activities of the first group of enzymes, represented by LIS and SAMT, increase in maturing buds and young flowers, peaking about 12 to 24 hours ahead of peak volatile emission. LIS and SAMT activities then decline in old (5-day) C. breweri flowers, but remain relatively high (40%–50% from the maximum level) even though emission of linalool and methylsalicylate has practically ceased. The activities of the second group of enzymes, represented by IEMT and BEAT, show little or no decline at the end of the lifespan of the flower, although, again, emission of methyleugenol, isomethyleugenol, and benzylacetate virtually cease. A minor difference in developmental profiles of the latter two enzymes is that IEMT levels peak on Day 1 of anthesis and stay stable afterward (Wang et al., 1997, Plant Physiology 114:213–221), whereas BEAT activity does not peak until the 4th day after anthesis (Dudareva et al., 1998, Plant Journal 14:297–304). Overall, these studies showed that scent production in C. breweri is a complex process that involves spatial and temporal patterns of regulation that are not necessarily identical for all of the enzymes involved.
In related genetic studies, researchers have begun to clone the genes which encode these floral scent biosynthetic enzymes and are beginning to uncover the underlying molecular mechanisms that control floral scent production and emission, and, in some instances, how particular varieties or species lose their ability to emit fragrance. Expression of genes encoding floral scent biosynthetic enzymes in the C. breweri flower is temporally and spatially regulated during flower development. Dudareva et al. (1998, Plant Journal 14:297–304) demonstrated that BEAT expression is tissue-specific; it is not expressed at detectable levels in leaves, and that among floral organs, the bulk of the BEAT MRNA transcripts are found in the petals. Similarly, Dudareva et al. (1996, Plant Cell 8:1137–1148) reported that LIS expression is most abundant in the petals, stigma, style, and is not found in the vegetative parts of the plant. The mRNA's encoding LIS, IEMT, and BEAT are first detected in petal cells just before the flower opens, and their levels increase until they peak at or around anthesis and then begin to decline (Dudareva et al., 1996, Plant Cell 8:1137–1148; Dudareva et al., 1998, Plant Journal 14:297–304; Wang et al., 1997, Plant Physiology 114:213–221). For all of these three genes, peak levels of the mRNA's occur 1 to 2 days ahead of the peaks of enzyme activity and emission of the corresponding compound. These gene expression results taken together suggest the presence of a common regulatory mechanism for floral scent biosynthetic genes whose mRNA levels peak at or around anthesis.
Overall, the data show that a good positive correlation exists between the amount of mRNA, the amount of protein and enzymatic activity for each of these enzymes, and emission of the corresponding component up to the second or third day post-anthesis. But beyond that point, the levels of scent enzymes remain relatively high despite declining levels of the corresponding mRNA and also without the concomitant emission of volatiles (Dudareva et al., 1996, Plant Cell 8:1137–1148; Dudareva et al., 1998, Plant Journal 14:297–304). These results also indicate that in C. breweri flowers, scent compounds are synthesized de novo in the epidermal cells of organs from which they are emitted (primarily the petals). Thus, those investigators concluded that the levels of activity of enzymes involved in scent production are regulated mainly at the mRNA levels at the site of emission.
The causes and consequences of appreciable levels of activity of biosynthetic enzymes in old flowers, without concomitant emission of the volatile products, were unknown. Nonetheless, the hereinbefore discussed investigators advanced several hypotheses to explain this result. First, they thought that it was possible that the biosynthetic pathways in which these enzymes participate are blocked elsewhere. Second, they thought that another possibility was that the products of the reactions catalyzed by these enzymes are required for processes other than scent emission in the flowers. Indeed, it has been found that the flowers of many species accumulate glycosides of scent compounds as they age. Such non-volatile glycosides are also sometimes found in buds, and were therefore originally hypothesized to be obligatory “scent precursors.”However, closer examination has shown that, in most cases, an increase in emission of a particular volatile is not accompanied by a corresponding decrease in levels of the glycoside of this volatile, as would be expected by this hypothesis. The increased synthesis of such glycosides as the flowers age may account for the cessation of scent emission, although the specific roles of such glycosides in the flower remain to be determined. Finally, they thought that a third possibility was that as the flower ages, substrates may be diverted to other compartments and are not accessible to the scent biosynthetic enzymes. Whatever the explanation, it was abundantly clear that high levels of activity of biosynthetic enzymes without concomitant emission of the volatile products could be found in disparate metabolic pathways (e.g., BEAT in the phenylpropanoid pathway and LIS in the monoterpenoid pathway) within the same flower. According to these investigators, these observations suggested the presence of a common, globally-aoting regulatory mechanism for control of floral scent emission.
Biochemical and molecular analysis of scent production in other flowers from the Clarkia genus have yielded some early insights into the underlying basis for scent production. The genus Clarkia, which is subdivided into eight sections, is a member of the evening primrose family and contains 44 species. With the exception of the moth-pollinated C. breweri, all other species of the genus have essentially nonscented flowers that are pollinated mostly by bees. The flowers of C. breweri, a species believed to have evolved recently from the nonscented C. concinna (the only other member of section Euchardium), emits a relatively simple mixture of monoterpenoid and phenylpropanoid compounds, but primarily the monoterpenoid, linalool. Dudareva et al. (1996, Plant Cell 8:1137–1148) were able to demonstrate that a large increase in, and a wider distribution of, LIS activity in C. breweri flowers as compared to C. concinna flowers. These observations did not reveal whether such changes were brought about by changes in the level of LIS gene transcription or by changes at subsequent steps controlling gene expression. In later studies, these questions were answered as RNA gel blot analysis revealed that low levels of LIS transcripts were detected only in the stigma of C. concinna flowers, and no transcripts were detected in the petals, styles, stamens, or sepals. Moreover, no LIS protein could be detected in any C. concinna floral organ. Taken together, these results demonstrated that the level of LIS protein is tightly correlated with the steady state levels of LIS mRNA in C. concinna, and the very low levels of both help explain the low levels of linalool emitted from C. concinna. Thus, these investigators concluded that the major regulatory mechanism(s) for biosynthesis and emission of floral scent in Clarkia flowers (both breweri and concinna) were found at the transcriptional and translational levels of the floral scent biosynthetic enzymes themselves.
In connection with their research on this invention, the present inventors hypothesized that another possible, though yet undiscovered, explanation for a reduction in or lack of floral scent might be due to inadequate levels or inaccessible pools of floral scent precursors. Although the identity of floral scent precursors is known in some instances (e.g., GPP, FPP, (iso)eugenol, benzyl alcohol, salicylic acid), there is a large body of unknown facts concerning these precursors. In most cases, the complete biochemical pathway(s) leading to the floral scent precursors are unknown. Also, the size of the pools of the precursors is unknown as well and is often difficult to quantify, due in part to the difficult and complicated assays that are required for analysis. Finally, even if floral scent precursors are detected, that still does not address the question as to the site of biosynthesis within the plant. For example, the detection of benzyl alcohol in C. breweri flower petals does not fully guarantee that this floral scent precursor was synthesized in situ, but rather could have been transported to the petals from the sepals, or the foliage, or even the roots. Thus, there exists a myriad of questions about the location(s) of intracellular sites for biosynthesis, identity of metabolic pathways involved, plant tissue sources, and regulatory steps for floral scent precursor biosynthesis in plants.
The present invention arose as the result of research conducted by the inventors to determine whether the emission of floral scents from plants could be modified by manipulating the supply of floral scent precursor compounds to plants. As a result of this research, the inventors have discovered that by supplying floral scent precursors to cut flowers, they have been able to dramatically modify the floral scent emission pattern from cut flowers. Moreover, the inventors have discovered that the modified emission pattern is dependent upon the presence of the floral scent precursor. These discoveries have also been extended to include a potted flowering plant in which the floral scent precursor compound has been exogenously supplied as a spray application. Moreover, the inventors have further discovered that the emission of volatile floral scent compounds which are seemingly unrelated to the floral scent precursor supplied are also altered and modified by a yet-unknown cellular mechanism. Finally, the inventors have demonstrated that the presence of a floral scent precursor can negate the adverse effects that certain climatic conditions (e.g., refrigerated temperatures and an extended period of darkness) can impose upon floral scent emission from cut flowers. Taken together, the inventors have discovered that the multi-component floral scent emission pattern of a flower can be maintained, enhanced or modified by supplying a single floral scent precursor to the plant.